The central objective of this proposal is to precisely define viral and cellular determinants of reovirus entry into cells. Mammalian reoviruses are non-enveloped viruses that enter cells by receptor-mediated endocytosis. To gain entry into cells, reovirus requires distinct receptors for attachment and internalization, endosomal acidification, and proteolysis of viral outer-capsid proteins. However, the dynamic mechanisms underlying the reovirus entry process are not understood. Four specific aims are proposed to elucidate mechanisms that promote reovirus entry. In Specific Aim 1, the capacity of RGD-binding integrins to serve as reovirus internalization receptors will be tested using RGD peptides, integrin-specific antibodies, integrin-deficient cells, and purified integrin heterodimers. Cellular proteins that interact with reovirus internalization receptors will be identified by immuno-affinity purification. In Specific Aim 2, mutations in reovirus genes that confer resistance to inhibitors of endosomal acidification will be selected and characterized. Functional domains in the viral outer capsid that regulate disassembly will be defined using viral particles recoated with outer-capsid proteins that display mutant disassembly phenotypes. In Specific Aim 3, three-dimensional structures of reovirus-integrin complexes and mutant viruses with entry-enhancing mutations will be determined using cryo-electron microscopy. The structure of major outer-capsid protein sigma3 derived from a protease-hypersensitive reovirus mutant will be determined by X-ray crystallography. In Specific Aim 4, an inhibitor of endocytic cathepsin activity expressed in mutant cells selected during persistent reovirus infection will be identified using cathepsin B as an affinity ligand. The role of endocytic proteases cathepsin B and cathepsin L in reovirus pathogenesis will be defined using genetically altered strains of mice. This research will reveal how viral and cellular proteins cooperate in a highly ordered biochemical pathway that culminates in reovirus entry. These studies may illuminate new targets for therapy against pathogens that use the endocytic pathway to parasitize host cells.